Recent studies have demonstrated the involvement of a group of zinc-dependent endopeptidases, called matrix metalloproteinase (MMPs), belonging to the matrixin family in the vascular alteration of cardiovascular pathologic conditions.
Venous hypertension is at the basis of several pathologies related to disorders of macro and micro circulation disorders and causing infiltrations of leucocytes in the endothelium, deterioration of the cardiac valves and finally providing the remodeling/rearrangement of vein wall, which can provide a reflux, or formation of varicose veins and derma pathologies as described by Bergam J. et al. in Ann. Vasc. Surg. 21 (2007), 260-266. Raffetto J D. et al. in Thromb. Res. 123 (2009) S66-S71 describe that the modifications observed during the progression of chronic venous pathology may be associated to an alteration of the hematic balance between the level of matrix metalloproteinase (MMPs) and their tissue inhibitors (TIMPs).
Both MMPs and TIMPs, being involved in the physiologic remodeling of extracellular matrix, play important role also in the cellular communication.
High MMPs concentrations have been found in different pathologies as reported by Mannello F. et al. in Curr. Cancer Drug Targets 5 (2005), 285-298. Among MMPs, MMP-9 (Gelatinase B, EC3.4.24.35 of 92, 130 and 225 KDa) are involved in several pathologies, comprising neoplastic pathologies suggesting that their presence as circulating enzyme and their increase are related to the progression of different cancer types, such as in breast, lungs, ovary and prostate cancer and melanoma, as described by Rahko E. et al. in Tumor Biol., 30 (5-6) (2009), 574-64.
In vascular pathological conditions, also caused by diabetes, it is clearly showed the pathogenic role of MMPs. Galis Z. S. et al. in Circ. Res. 90 (2002), 251-262, Kadoglou N. P. et al. in Angiology 56 (2005), 173-189, Derosa G. et al. in Diabetes Metab. 33 (2007), 129-134 and Tayebjee M. H. et al. in Diabetes Care 27 (2004), 2049-2051 describe an increased level of circulating MMP in diabetic patients.
Raffetto J D. et al. in Biochem. Pharmacol. 75 (2008), 346-359, demonstrate the importance of MMPs in vascular disease, reporting that the increased extracellular matrix degradation (ECM) in venous wall, is involved in the early steps of venous relaxation, formation of varicose veins, dermal modification and finally leading to the formation of venous ulcers.
Among the several pathological mechanisms linked to the increased expression of MMPs, the interactions leucocytes-endothelium are important and represent a possible objective for the pharmacological treatment. Mannello F. et al. describe in Arterioscler. Thromb. Vasc. Biol. 28 (2008), 611-614, that leucocytes and platelets contain higher amounts of MMPs, which can be released into the extracellular milieu consequently to the leukocyte activation/degranulation or during platelet aggregation. Furthermore, MMP-9 is released from platelets and leucocytes after a stimulus such as the reactions induced by the coagulation process.
It is known that the plasmin, activated during in the coagulation phase, is in turn a MMPs activator.
Among proteolytic enzymes present in human walls and potentially associated to the cancer invasions, the MMPs have been demonstrated to be involved in the capacity to degrade almost proteins of the interstitial matrix and basal membranes, allowing to the cells to diffuse and infiltrate in the neighboring walls.
Several compounds, such as growth factor receptors of adhesion cellular molecules, chemokines, cytokines, apoptotic ligands and angiogenic factors may interact with MMPs, modifying both its expression and activity.
These proteolytic enzymes act on many bioactive substrates being involved in the neoplastic development steps such as growth of primary tumor, angiogenesis, extravasation or intravasation of the neoplastic cells, migration, invasion of metastatic cells in the secondary organ, beginning and maintenance of the tumor growth.
Several clinical studies have been performed in both animal and human tumoral models to evaluate the importance of the reduction of circulating matrix metalloproteinase. The obtained results have lead to develop synthetic inhibitors as described by Ramnath N. et al. in Curr. Oncol. Rep. 6 (2004), 96-102.
The main inhibitors studied comprise mimetic peptides and non mimetic peptides of collagen, of tetracyclines and bisphosphonates derivatives.
Clinical trials executed with the available compounds have shown that prolonged treatments cause muscular-skeletal pain and inflammation. Although these events showed to be reversible and the patients started again the treatment after a short suspension period, the unexpected adverse events have limited the administration dosages.
Moreover, when the clinical trials for the treatment of neoplastic diseases were extended to a greater number of patients, besides serial toxicological problems due to the cytostatic effects, no significant therapeutic benefit was observed, as described by Mannello F. et al. in Curr. Cancer Drug Targets 5 (2005), 285-298.
Some MMPs inhibitors studied in clinical trials are Batimastat, Marimastat, Prinomastat, BAY 12-9566, CGS27023A and derivatives of tetracyclines.
Batimastat, BB-94 (WO 90057191), is a hydroxamic acid derivative, which mimes the peptidic structure of natural substrates. Batimastat is a powerful inhibitor of MMPs and has been the first inhibitor of MMP used in clinical trials, but it has shown low selectivity caused by low solubility and absorption. Furthermore, Batimastat had a lower compliance, being administrable only by injection in the pleural or abdominal space. The Phase III clinical trials were almost immediately suspended for serious side effects, such as strong local inflammatory tissue reaction, nausea, abdominal pains as reported Tu G. et al. in Curr. Med. Chem. 15 (2008), 1388-1395.
Marimastat, BB-2516 (WO 9402447), is a hydroxamic acid derivative with a structure similar to Batimastat, but with a higher solubility and then easier absorption by oral administration. Marimastat as well as Batimastat has low specificity and it was toxic in a percentage of about 30% of all treated patients, showing muscular-scheletal pains and rigidity, which starts in the little hands knuckle and moves up to arms and shoulders, especially around the tendinous insertion points, fibrosis and necrosis of the periarticular walls of ankle and knee and gastric disorder associated to weight loss as described by Vihinen et al. in Int. J. Cancer 99 (2002), 157-166.
Prinomastat, AG3340 (WO 90720824) is a hydroxamic acid derivative, selective against some MMPs involved in the tumoral invasion and metastasis. During the clinical trials, side effects occurred in the joint of shoulder, knee and hands, appeared and the treatment was immediately suspended, as described in Ramnath N. et al. in Curr. Oncol. Rep. 6 (2004), 96-102.
BAY 12-9566, (U.S. Pat. No. 4,705,798) is a butanoic acid derivative and in several clinical trials it has showed toxicity, represented by thrombocytopenia, anemia, increase of hepatic enzymes and bilirubin, nausea, tiredness and headache as described by Nelson A. et al. J. Clin. Oncol. 18 (2000), 1135-1149.
CGS27023A, (U.S. Pat. No. 5,455,258) has reported toxic effects with a wide cutaneous irritation, myalgia and arthralgia.
Also tetracyclines derivatives have shown severe adverse events during the clinical trial, such as tiredness, mental confusion, nausea, vomit, cutaneous phototoxicity, pancreatic enzymes increase, which limited the administrable dosage, as described by Hidalgo M. et al. in J. Natl. Cancer Inst. 93 (2001), 178-193.
Zymography and reverse zymography are techniques currently used for the MMPs and TIMPs analysis in biological samples. All types of substrates zymography originate from the gelatin zymography method. The techniques are the same except that the substrate differs depending on the type of MMPs or TIMPs. In these techniques, proteins are separated by electrophoresis under denaturing, not reducing conditions, in the presence of sodium dodecyl sulphate (SDS). Gelatin-zymography is a technique used mainly for gelatinase MMPs determination and it is extremely sensible to detect MMP-2 and MMP-9 at concentration of few picograms. It should be considered that other MMPs, such as MMP-1, MMP-8 and MMP-13 can also degrade the substrate.
The technique is based on electrophoresis separation, in polyacrylamide gel with a specific substrate; SDS denatures the MMPs which become inactive and after the electrophoresis migration, the SDS is removed from the gel in a suitable buffer, which restores the MMPs structures and functions. After that, if the gel is incubated, the activated gelatinases digest gelatine which is converted into low molecular weight peptides, that are removed by washing as described by Mannello F. et al. in Clin. Chem. 49 (2003), 339-34.
The gels are placed in a development solution, for example comprising Coomassie, and then destained with solutions comprising methyl or ethyl alcohol and acetic acid. The stained zone of the gel are due to the presence of not-degraded gelatine, while the not stained zone are due to the enzymatic activity of MMP. The enzymatic activity results proportional to the extension of the clear bands, thus it can be analyzed and quantified with densitometer instruments equipped with imagine analyzer.
Zymograms provide specific and distinct information on MMPs and in particular on MMP-2 and MMP-9, because the two enzymes can be separated on the basis of the different electrophoresis mobilities, which depend on their molecular weights. For this purpose, suitable molecular weight standards of corresponding to pro-MMP-2 72 kDa, and complex of pro-MMP-9 and MMP-9 at 92, 130 and 225 kDa are placed for comparison in the same gel.
Mannello F. et al. in Clin. Biochem. 41 (2008), 1466-73 studied the effect of the addition of high-molecular-weight lithium heparin to peripheral blood samples, for evaluating the effect of HMWH on MMPs and TIMPs expression in blood. The described effect was that HMWH had both direct and indirect effects altering the quantity of circulating MMP and TIMP and increasing the release of TIMP-2. The objective of this publication was essentially to describe the effect of a high-molecular-weight heparin (HMWH) in the MMPs expression in blood manipulation in order to standardize the procedure of blood sample handling, in a method of measuring MMPs. In this publication there are no references and/or suggestion regarding pharmacological uses of heparin. Moreover, it is not reported the lithium heparin concentrations, at which the effect is observed. As a consequence, it is not possible to know if the concentration of lithium heparin, which provided the effect observed, might be therapeutically useful, being well known the high risk of bleeding associated to HMWH. Nevertheless, being known that heparins are commonly administered by injection any possible heparin use done to the purpose of altering MMPs plasma concentrations, has to be supposed following the same route.
The effect of heparin on the MMP-2 and TIMP-2 mRNA expression is also reported by Caenazzo I. et al. in Nephrol. Dial. Transplant 2001, 16, 1769-75.
Therefore there is still the need to find active ingredients for medicine preparations, useful in the reduction of blood circulating MMPs concentration in the intent of treating all the pathologies wherein MMPs are involved, as pathogenesis of inflammation, infective or neoplastic or cardiovascular disease. Among these pathologies, are comprised for example, pathologies derived by diabetes, varicose veins, chronic venous insufficiency (CVI), atherosclerosis cardiac break after myocardium infarct, abdominal aortic aneurysm, pulmonary pathology, increase and progression of the tumor, growing in the primary tumor, abnormal angiogenesis, extravasation and intravasation of the neoplastic cells.
Vascular diseases are selected in the group of varicose veins, human atherosclerosis, cardiac break after myocardium infarct, abdominal aortic aneurysm.
Moreover there is the need to obtain medicines, without any side or toxic effect at therapeutically effective dosage, acceptable by the patients and administrable by oral route, intramuscular or intravenous injection for the treatment of all pathologies wherein MMPs are involved.
It has been unexpectedly found, and it is an object of the present invention, that sulodexide, or one of its component, in particular dermatan sulfate obtained by sulodexide (SDX-DS) or heparin (SDX-HEP) obtained by sulodexide, inhibit and/or reduce the level of circulating MMPs.
Sulodexide is a natural mixture of natural glycosaminoglycans, GAGs, extracted from intestinal mammalian mucosa, as described in U.S. Pat. No. 3,936,351, comprising a mixture of fast-moving heparin (SDX-HEP), with average molecular weight of 7 kDa, and dermatan sulfate (SDX-DS), with average molecular weight of 25 kDa. Other possible components comprised in sulodexide are chondroitin, heparin sulfate. Sulodexide has to be considered as a unique active ingredient, different from other glycosaminoglycans, comprising about 80% in weight of SDX-HEP and about 20% in weight of SDX-DS. These components have not been isolated until now and the components of sulodexide are determined with analytical method as gel permeation chromatography and electrophoresis.
It is known that sulodexide shows high affinity for antithrombin III and for Heparin-cofactor II, inhibits the factor Xa and thrombin, activates tissular plasminogen and reduces the fibrinogen level as described by Ofosu F. A. in Semin. Thromb. Hemost. 24 (1998), 127-38 and by Ceriello A. et al. in Diab. Nutr. Metab., 6 (1993), 1-4.
Since, it is known that the plasmin activation from plasminogen is one of the local activation mechanism of MMPs, the known properties of sulodexide lead to conclude that this compound would have increased the concentration of circulating MMPs.
The present invention describes sulodexide or one of its components, in particular SDX-DS or SDX-HEP, to inhibit and/or reduce circulating MMPs, and in particular MMP-9, said also gelatinase B, involved in a number of diseases, in particular, an infection or a neoplastic disease, vascular disease characterized by a high level of MMPs, gastrointestinal ulcers, increase and progression of the tumor, growing of the primary tumor, altered angiogenesis, extravasation and intravasation of the neoplastic cells and combination thereof. As to vascular disease characterized by a high level of MMPs, the present invention, in particular relates to chronic venous insufficiency (CVI); varicose veins, cardiac break after myocardium infarct, abdominal aortic aneurysm, the venous relaxing, pulmonary pathology, and neoplastic pathologies.
Another unexpected and important aspect of the present invention is the dermatan sulfate (SDX-DS) and the low molecular weight heparin (SDX-HEP) isolated from sulodexide, the process to obtain them and their use to inhibit and/or reduce the release of MMPs in vitro and in vivo, alone or in their mixture.
This result is indeed unexpected, because in literature, for example in Cell Biol. Int. 2003, 27, 779-84, Isnard N. et al. describe that the dermatan sulfate activates the expression of MMP-2 and MMP-9.
The surprising characteristics of the SDX-DS, have been demonstrated comparing it with commercial dermatan sulfate, and results are shown in Examples 2-9.
This evidence makes sulodexide indeed a unique compound, different from any other glycosaminoglycans, which might be prepared with low molecular weight heparins and dermatan sulfates, available in the market, for the treatment of all pathologies wherein MMPs are involved.
Another aspect of the present invention is the SDX-DS isolated from sulodexide.
Another aspect of the present invention is the SDX-HEP isolated from sulodexide.
Another particular aspect of the present invention is the dermatan sulfate obtained from sulodexide through purification process, such as in affinity chromatography with AT III functionalized medium.
Another particular aspect of the present invention is the low molecular weight heparin obtained from sulodexide through purification process, such as in affinity chromatography with AT III functionalized medium.
Another aspect of the present invention are pharmaceutical compositions comprising SDX-DS or SDX-HEP and their use as medicine.